Isolation and characterization of herpes simplex virus 1 primary enveloped virions
Padula, Maryn E.
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The life cycle of herpes simplex virus 1 (HSV-1) begins in the nucleus: the viral genome is replicated and packaged into a newly formed capsid; mature capsids with packaged viral genomes are termed nucleocapsids. Nascent nucleocapsids bud into the inner nuclear membrane and enter the perinuclear space as enveloped particles termed primary enveloped virions. These enveloped intermediate particles fuse with the outer nuclear membrane and the de-enveloped nucleocapsids enter the cytoplasm. Maturation is completed upon acquisition of a secondary envelope from the TGN or endosomal regions.;Primary enveloped virions have been widely studied by those interested in HSV-1 egress because these particles differ morphologically from the mature virus. However, the formation of the perinuclear HSV-1 particle is still poorly understood. The objective of this thesis was to characterize the protein composition of primary enveloped virions and then analyze individual protein components to determine their role in HSV-1 assembly. Our laboratory developed a novel biochemical approach to isolate primary enveloped virions from cells. The method was tested to determine its integrity and then mass spectrometry was used to identify protein components. This analysis led to the most comprehensive list of primary enveloped virion protein components to date and included many known proteins as well as several novel candidates.;The novel identifications from the proteomics analysis included a host protein, annexin A2, and a viral tegument protein, VP22. This thesis will show that annexin A2, a protein known to associate with membranes, is packaged into primary enveloped virions, but under our cell culture conditions plays a non-essential role in HSV-1 virion production. The tegument protein VP22 has been extensively studied yet its function is still unknown. Multiple forms of VP22 are found during an HSV-1 infection, and these forms were further examined. While several forms are associated with nuclear membranes, only a single species is packaged into primary enveloped virions, the same form found in the mature virus.;Isolation of primary enveloped virions and elucidation of their protein components provide a new approach to studying HSV-1 egress and assembly and will provide new insights into HSV-1 biology.